Vascular smooth muscle (VSM) and endothelium play primary roles in pregnancy-associated vascular adaptations. Peptides of the calcitonin gene-related peptide (CGRP)-family-CGRP, adrenomedullin (AM), and intermedin (IMD)-have been shown to play significant roles in these adaptations. CGRP, AM, and IMD share common receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)1, 2, and 3, in different combinations in exerting their biological functions. During the previous funding periods, we have demonstrated that CGRP, AM, and IMD play critical roles in regulating pregnancy-associated vascular adaptations, utero placental functions, and fetal growth. Interestingly, the effect of these peptides varied depending on the vascular bed and between VSM and endothelial cells. Our new preliminary findings suggest variable associations and competitive interactions of RAMPs with CRLR could contribute to the observed differences in peptide-induced relaxation between mesenteric artery (MA) and uterine artery (UA). Tumor necrosis factor- (TNF) caused differential and selective decreases in RAMPs or CRLR in VSM and endothelial cells. We hypothesize that differences in vasorelaxations to CGRP, AM, and IMD in both MA and UA are determined by differential association and competitive interaction of different RAMPs with CRLR and that these associations and interactions are cell-specifically downregulated by TNF. We will utilize intracellular cAMP measurements, shRNA-mediated receptor knockdowns, novel proximity ligation assay (in situ method to quantitatively detect CRLR and RAMP protein proximity associations at the cell membrane level), and functional vascular reactivity studies to test our hypotheses. Four specific aims are proposed. Specific Aim 1: To investigate molecular mechanisms of action for 3 peptides of the CGRP family on MA VSM cells. We will determine competitive association of specific RAMPs with CRLR and cAMP generation in MA VSM cells and vasorelaxation responses to peptides in MA rings with knockdown of specific RAMPs. Specific Aim 2: To assess molecular mechanisms of action for 3 peptides of the CGRP family on UA VSM cells. We will determine association of RAMPs with CRLR and cAMP generation in UA VSM and relaxation response to peptides in UA rings with knockdown of specific RAMPs. Specific Aim 3: To investigate expression and association of RAMPs with CRLR in endothelial cells and assess effects of 3 peptides on eNOS expression and phosphorylation and NO production. Specific Aim 4: To determine if TNF alters expression and molecular interactions of RAMPs and CRLR and, therefore, actions of 3 peptides in VSM and endothelial cells. These findings will significantly advance our knowledge of molecular mechanisms underlying CGRP family peptide-induced vascular adaptations and offer important original insights into the possible mechanisms of vascular dysfunction in pathological pregnancies.